Agonists and Receptors
Cells receive and respond to signals in their environment. Such signals are commonly transmitted to cells by signaling molecules, such molecules also commonly being produced by cells. One type of signaling molecule interacts with, or binds to, cellular receptors. When a signaling molecule binds to a receptor, processes in the target cell that lead to a biological response are initiated. Such processes normally constitute intracellular reactions of various signal transduction pathways. The endpoints of such pathways are changes in a variety of cellular behaviors or responses, including metabolism, differentiation, proliferation, cell death and others. Molecules that interact with receptors are commonly referred to as ligands. Ligands that initiate or affect such cellular behaviors through interaction with receptors are called agonists.
There are many different types of signaling molecules that have agonist activity, examples of which include hormones, growth factors, cytokines, chemokines, neurotransmitters, and the like. Other signaling molecules include steroids, retinoids, thyroxins, prostaglandins, leukotrienes, and others. Substances such as toxins, synthetic molecules and certain drugs can also exert their effects through interaction with receptors. The cellular receptors with which these ligands interact can be intracellular or extracellular. Normally, there is some specificity in interaction of a ligand with a receptor.
Ligands can interact with receptors in a variety of mechanisms. For example, the stoichiometry of the interaction can be one ligand interacting with one receptor. Alternatively, receptors may be able simultaneously to interact with more than one ligand. Ligands may be able to interact with more than one receptor during a binding event.
Prolactin is an example of a ligand that can interact with more than one receptor during a single interaction. Prolactin is an approximately 23,000 Dalton protein that may be found in glycosylated forms. Prolactin is a cytokine in the same family as growth hormone.
Prolactin's tertiary structure has been determined by nuclear magnetic resonance and is an up-up-down-down four-helix bundle topology (FIG. 1). This general structure is similar to those observed for other members of this protein family, including growth hormone and placental lactogen. This topology has also been described as a four-helix bundle scaffold or as a bundle of four alpha helices.
Prolactin has two receptor-binding sites or surfaces. Site 1 is composed of portions of helices 1 and 4. Site 2 is located around the cleft defined by helices 1 and 3. The amino acids that lay within these surfaces form two distinct atomic topologies that bind the prolactin receptors.
The biological responses initiated by prolactin are mediated by its interaction with the prolactin receptor. Prolactin receptor is a Type I cytokine receptor, with a cytoplasmic domain of variable length, a single transmembrane domain and an extracellular domain that interacts with prolactin. The prolactin receptor also bears a high degree of homology to the growth hormone receptor. In fact, primate growth hormones and placental lactogens can bind to and activate the prolactin receptor. In contrast, however, prolactin does not bind to the growth hormone receptor.
Current insights into the molecular mechanisms by which prolactin binds to and activates its receptor are based, at least in part, on the more extensive studies of the interactions of growth hormone with its somatotrophic receptor. Published studies demonstrate that growth hormone activates the growth hormone receptor through a sequential receptor dimerization mechanism (i.e., form ternary complexes or also called ligand-induced receptor oligomerization). In this mechanism, site 1 on the hormone first binds to one molecule of receptor and then site 2 on the hormone binds to a second molecule of receptor.
Supportive evidence for the two-site binding includes the following: (i) Bivalent anti-growth hormone receptor antibodies can activate the receptor, while monovalent Fab antibody fragments cannot. (ii) Crystal structures of growth hormone bound to the extracellular domains of growth hormone receptor or prolactin receptor demonstrate the interaction of two receptor monomers with a single growth hormone molecule through non-symmetrical sites on growth hormone. (iii) In vivo growth hormone display self-antagonism in response to increasing concentrations of growth hormone. The growth responses to low concentrations of growth hormone increase with dose, but high concentrations of growth hormone are inhibitory. A smaller amount of information is available for the interaction of prolactin and the prolactin receptor, but these data also are consistent with sequential receptor dimerization mechanism. As with growth hormone divalent antibodies activate the prolactin receptor while monovalent antibodies do not. Prolactin also antagonizes its activity at high concentrations.
It is thought that prolactin-induced dimerization of prolactin receptor stimulates the JAK-STAT kinase signal transduction pathway to activate gene expression. The cytoplasmic tail of the prolactin receptor does not possess kinase activity. However, ligand-induced dimerization of prolactin receptor leads to the association of the JAK2 kinase with the cytoplasmic portion of the prolactin receptor and results in phosphorylation of both the receptor and JAK2. Phosphorylation of the receptor then leads to association of the transcription factors STAT 1, 3, 5a and 5b with the receptor, via their SH2 domains. The latter association then leads to the phosphorylation of the STAT proteins by the JAK2 kinase. This phosphorylation event is required for subsequent dimerization of the STAT transcription factors, transport to the nucleus and transcriptional activation. STAT5a and STAT5b have been shown to be crucial for the development of the mammary gland. Gene knockouts of these transcription factors mimic many of the features of prolactin receptor knockouts. In particular, STAT5b phosphorylation seems to correlate most closely with proliferative effects in cells.
In addition to the scheme described above, several reports have demonstrated that prolactin can activate elements of the mitogen-activated protein kinase (MAPK) pathway, including the src, the src homology, flyn, Raf-1, and MAP kinases. It is becoming clear that considerable crosstalk exists between the JAK-STAT and MAPK signaling pathways. Some evidence suggests that the STAT proteins can be phosphorylated and activated by both pathways, although the mechanism of activation may be distinct in each case. Nevertheless, phosphorylation of the STAT proteins appears to be an intercellular surrogate marker for the biological effects of prolactin.
The pituitary, placenta, and other tissues of mammals produce prolactin. Prolactin interacts with prolactin receptors, which exist in a variety of tissues including the breast, liver, prostate, kidney, and cells of the immune system. A widely studied biological response action of prolactin is in the development and lactation of the epithelial cells of the breast (mammary tissue). During lactation, lactating epithelial cells of the breast are dependent on prolactin. Prolactin also affects growth, development, and/or survival of tumors of the breast or mammary gland.
Human prolactin is increasingly associated with the development and growth of human breast tumors. Most breast tumors develop from the mammary epithelial cells that produce milk. These tumor cells possess prolactin receptors and produce prolactin. Therefore, it appears that these tumors have acquired an autocrine system: they make their own prolactin and release it into the extracellular space where it binds prolactin receptors of the tumor.
Other tumors appear to be prolactin-dependent or at least prolactin-responsive. For example, prolactin has been implicated in normal prostate development and prostatic hyperplasia and hypertrophy.
Breast cancer and prostate cancer are the second leading causes of cancer-related deaths among women and men, respectively. Together, these two tumor types were responsible for more than 360,000 new cases and 73,000 deaths in the United States during the year 2000. Few therapeutic compounds increase long-term survival or reduce morbidity. For breast cancer, surgery and/or radiotherapy are the mainstays of treatment of localized disease, with cyclophosphamide, doxorubicin, 5-fluorouracil, and paclitaxel commonly used separately or in combination chemotherapeutic regimens. In addition to these now common therapeutic strategies, beneficial effects have been observed with the anti-estrogens and tamoxifen (a partial agonist/partial antagonist of the estrogen receptor), but such responses occur only in patients whose tumors express sufficient concentrations of estrogen receptors.
The situation for prostate cancer is even worse. Prostatectomy and/or radiotherapy are most commonly used to control local disease. However, metastatic disease has proven refractory to nearly all chemotherapeutic regimens tested. The only consistently successful chemotherapeutic approach identified is the complete inhibition of androgen action, which may require both the ablation of testicular androgen synthesis and the administration of anti-androgens to block the effects of androgens secreted by the adrenal glands.
Antagonists
When acting as an agonist, ligand binding to its receptor produces a biological response as described above. Molecules exist that interfere with the ability of ligands to produce their biological responses. Such molecules are called antagonists. Antagonists are substances that suppress, inhibit, or interfere with the biological activity of a native ligand (e.g., a signaling molecule). Antagonists can function in a variety of ways. One way in which antagonists can function is by binding or interacting with a receptor at the same site on the receptor to which an agonist binds. In this case, binding of the antagonist to the receptor inhibits the ability of the agonist to bind to the receptor. Functioning of an antagonist in this way is called competitive antagonism. Another way in which antagonists can function is by binding or interacting with a receptor at a different site on the receptor to which the agonist binds. In this case, binding of the antagonist to the receptor can prevent agonist binding or, if the agonist does bind, transmission of its signal to the signal transduction pathway is inhibited. Functioning of an antagonist in this way is called noncompetitive or uncompetitive antagonism.
Investigators have realized the therapeutic potential of antagonists in certain circumstances. For example, an effective prolactin antagonist would block or inhibit the ability of the body's own prolactin to cause a biological response. Such prolactin antagonists can be used as prophylactic or therapeutic agents for breast cancer, prostate cancer, other prolactin dependent tumors, or can be given to females after the birth of a child for the purpose of reducing or suppressing lactation.
Prolactin antagonists, specifically prolactin variants that have antagonist activity, have been described in the prior art. In these compounds prolactin has been modified by replacing an amino acid within one of the two receptor-binding surfaces of human prolactin (i.e., within site 1 or site 2) with an amino acid that blocks receptor binding (e.g., replacement of a small amino acid with a bulky amino acid) through that site on the ligand. Alternatively, prolactin has been modified by making mutations within amino acids that form the “scaffolding” that holds the global structure of the protein together—such mutations also disrupt the structures of site 1 and/or site 2. In one type of prolactin antagonist, the structure of the site 2 receptor-binding surface is affected to physically block the binding of prolactin to the prolactin receptor at site 2. The logic approach is that by presenting prolactin antagonists to tumor cells that bind but cannot dimerize receptors, prolactin receptors would be bound but not activated. With sufficient receptor binding by antagonists, insufficient receptors would be available for the endogenous agonist to initiate a biological response.
This approach has been marginally successful, producing an antagonist that retains approximately 1% agonist activity. See U.S. Pat. No. 6,429,186, to Fuh et al. This approach produces a less than desirable therapeutic agent, however, because treatment with antagonist concentrations sufficient to interfere with the autocrine prolactin (i.e., high concentrations), produce significant agonist activity. For example, if an effective pharmacological concentration of an antagonist requires a 100-fold excess concentration of antagonist over the endogenous agonist, then retention of 1% agonist activity in the drug will defeat its purpose because the drug's agonist actions will be significant at the required concentrations.
Another approach to creating prolactin antagonists is described in U.S. Patent Application Publication No. 2001/0036662 to Walker. This approach also involves mutation of amino acids believed to be directly involved in binding. However, like the Fuh approach (U.S. Pat. No. 6,429,186), this approach yields a product that exhibits agonist activity—in this case, about 10%. As noted above, this high level of agonist activity is unacceptable.
Therefore, there is a need for improved antagonists, in particular, better prolactin antagonists that efficiently block the activity of prolactin without providing undesirable agonist activity.